Applications in bioprinting are always climbing to new heights in terms of what parts of the body researchers can restore. Now, researchers at Utah University have developed a method for bioprinting ligaments and tendons out of the patient’s own cells. The process could be a breakthrough in the treatment of musculoskeletal tissue.

The process involves printing human tissue by taking stem cells from the patient’s own body fat. They take the fat and print it onto a layer of hydrogel to form a tendon or ligament. Then, they grow the cells in vitro in a culture before implanting them in a patient. The process will apparently allow patients to receive replacement tissues without additional surgeries and without having to harvest tissue from other sites.

The complexity of cells and tissues in ligaments and tendons form very intricate patterns. As a result, surgeons have a tough time reattaching these parts. The connective tissue comprises of tendons or ligaments but also parts that transition into bone cells to connect with the skeletal system. Researchers have tackled this problem with a technology that lets them precisely put cells where they need them. This is the major breakthrough of this whole experiment.

Developing Hydrogels

The bioprinter they used in the research was actually repurposed from other medical applications. The original Carterra microfuidic printer actually prints antibodies for cancer screening, but the researchers managed to modify it with special print head for the printer that can lay down human cells in a very controlled way. To properly print out the ligaments they first needed to conduct tests.

For the tests, they used genetically modified cells that glow a fluorescent color so that they could see the process in action. This served as a proof of concept for ligament, tendon and spinal disc printing but the team says the method’s applications could range to any sort of cell imaginable. Although, the current set-up is tuned to musculoskeletal applications, by customising the printhead and the type of printer, the same process allow for printing whole organs.

While printing a cell in isolation has proven easier, this is something new. The researchers have managed to print out a cellular structure that transitions from varying types of tissue. Thus, the research serves as a crucial step forward for understanding how to print complex tissue.

Tooth enamel protects teeth by providing a hard surface resistant to wear and tear, withstanding impacts without breaking over the lifetime of an organism. The Giant Panda has particularly clever tooth enamel, according to researchers, which can recover its structure and geometry to counteract the early stages of damage [Liu et al., Acta Biomaterialia(2018), https://doi.org/10/1016/j.actbio.2018.09.053]. The team from the Institute of Metal Research, Chinese Academy of Science, the University of Science and Technology of China, Lanzhou University of Technology, and the University of California Berkeley believe their observations could be replicated in the tooth enamel of all vertebrates, including humans, and inspire the design of artificial durable ceramics.

“Tooth enamel possesses an exceptional durability and plays a critical role in the function of teeth, however, [it] exhibits a remarkably low resistance to the initiation of large-scale cracks comparable to geological minerals,” points out Robert O. Ritchie, who led the study.

The ingenious design of the Panda’s tooth enamel, which has to withstand a daily diet of bamboo – a material of remarkable strength and toughness, comprises parallel microscale prisms made up of vertically aligned nanoscale fibers of the mineral hydroxyapatite embedded in an organic-rich matrix. When there is an impact on the enamel, a variety of different deformation mechanisms take place to mitigate the growth of small cracks and prevent the formation of large cracks.

“The tooth enamel is capable of partially recovering its geometry and structure at nano- to microscale dimensions autonomously after deformation to counteract the early stage of damage,” explains first author Zengqian Liu. “[This] property results from the unique architecture of tooth enamel, specifically the vertical alignment of nano-scale mineral fibers and micro-scale prisms within a water-responsive organic-rich matrix.”

Hydration plays a key role in the process. The viscoeleasticity of the organic-rich matrix surrounding the mineral prisms and fibers facilitates self recovery, while the presence of water decreases the width of any cracks that do form, with only a minor cost in terms of hardness.

“Our findings identify a novel means by which the tooth enamel of vertebrates develops an exceptional durability to accomplish its functionality,” says Liu. “The self-recovery process represents a new source of durability that differs markedly from the conventional protocol of fracture mechanics.”

As the architecture of the Panda’s tooth enamel is essentially similar to other vertebrates, the researchers believe that this self-recovery behavior is likely to occur in tooth enamel in general.

“Our findings also offer inspiration for the development of artificial durable, self-recoverable ceramic materials,” says Ritchie.

The team is hoping to develop tooth enamel-inspired self-recoverable durable materials by introducing shape-memory polymers at the interfaces of ceramics.

Dispersible electrodes based on gold-coated magnetic nanoparticles modified with DNA can detect microRNA in unprocessed blood samples at extremely low concentrations and over a broad range – a first for sensors of this kind. The devices, which have been tested on mice, can produce results in just 30 minutes and might be used to make a finger-prick test for early-stage cancer diagnosis.

“There are many microRNAs (short ribose nucleic acid sequences between 19 and 25 bases long) that are post-transcriptional gene expression regulators – that is, they can turn genes on and off,” explains John Justin Gooding of the University of New South Wales in Australia, who led this research effort. “The levels of these miRNAs are indicative of a range of pathologies, including cancers. If we could detect these RNAs in blood, where they circulate, we could make a finger-prick test as an early cancer diagnostic.”

Detection levels as low as 10 attomoles

“The problem is that the RNAs are found at very low concentrations of 10 femtomoles (fM) to 1 picomole (pM), so this is no easy task. Our new technique can detect levels as low as 10 attomoles (aM) and above 1 nanomoles (nM), so covering this entire range. What is more, it produces a result in just 30 minutes.”

Gooding and colleagues developed gold-coated magnetic nanoparticles (Au@MNPs) modified with DNA that is complementary to the miRNA they want to detect. “We call these magnetic nanoparticles ‘dispersible’ electrodes because they diffuse throughout the sample to capture the miRNA,” says Gooding. “When we then apply a magnetic field, these tiny electrodes reassemble to form a bigger electrode.

“When miRNA is bound to the DNA of the nanoparticles, the electrochemical current through the macro-electrode changes. The electrode measures this change and produces a signal,” he explains. “Since the nano-electrodes disperse throughout a sample, they capture nearly all the miRNA in it and the more they capture, the bigger signal. This is why our device is so sensitive.”

Fast response time

The sensor also has a fast response time since it makes use of an applied magnetic field to “bring back” all the captured miRNAs to the macro-electrode, he adds. “It can be likened to a hunter-gatherer that is on a motorcycle rather on foot. When sent out to find ‘food’, it covers more territory, so collects more food and brings it back faster.”

The technique is better than the current gold standard to profile miRNA, the real-time polymerase chain reaction (qRT-PCR), which does not work on samples of whole blood (it requires isolated and purified RNA). Although highly reliable, qRT-PCR is also labour-intensive and time consuming.”

“Our sensor is the first to be able to detect concentrations of miRNA from 10 aM to 1 nM in unprocessed blood samples,” Gooding tells Physics World. “We found that it can also distinguish small variations in miRNA concentrations in blood samples taken from mice with growing tumours.

“We believe that our work is an important advance for developing liquid biopsies for early cancer detection, that is before symptoms of the disease actually appear, and to monitor how well, or not, a treatment is working,” he adds.

Researchers at the University of New Hampshire (UNH) have created an easy-to-make, low-cost injectable hydrogel that could help wounds to heal faster, especially for patients with other health issues.

Wound healing can be complex and challenging, especially when a patient has other health obstacles that seriously impede the process. Often injectable hydrogels are applied to irregular shaped wounds, like diabetic ulcers, to help form a temporary matrix, or structure, to keep the wound stable while cells rejuvenate. The caveat is that current hydrogels are not porous enough to allow neighboring cells to pass through to help the wound mend.

“While valuable for helping patients, current hydrogels have limited clinical efficacy,” said Kyung Jae Jeong, assistant professor of chemical engineering at UNH. “We discovered a simple solution to make the hydrogels more porous and therefore help to speed up the healing.”

In the study, recently reported in ACS Applied Bio Materials, the researchers outline how they made a macroporous hydrogel by combining readily available gelatin microgels – hydrogels that are a few hundred microns in diameter – with an inexpensive enzyme called microbial transglutaminase (mTG). Gelatin was used because it is a natural protein derived from collagen, a protein found in connective tissue in the body such as skin.

Assembling these tiny microgels with mTG helped create a hydrogel with large enough pores for the neighboring cells to move into the wound for repair. In addition, this new injectable formulation allows for the slow release of protein drugs such as platelet-derived growth factor (PDGF) to aid wound healing. The researchers compared conventional nonporous hydrogels with the new macroporous hydrogels, and found a notable increase in the migration of tissue cells inside the hydrogel, which is the hallmark of wound healing.

Along with diabetic ulcers, the macroporous hydrogel could help with healing of wounds on the skin, cornea and internal organs during surgery, as well as having military implications.

Recent developments in bioprinting allow researchers to produce materials that can contain living cells within them. Often, these are bioinks or biolfilms that can carry complex biological data and become a complex printed structure. Now, researchers from multiple institutions have developed a gel that uses oxygen sensitive nanoparticles for the printing of complex biofilms and tissue-like structures. The finding allows the researchers to detect oxygen content and visually indicate it using sensors. This new venture has potential applications in biophotonics, biomedicine and biochemistry.

The study demonstrates how these bio-inks could use sensor nanoparticles for monitoring algal photosynthesis and respiration as well as stem cell respiration in bio-printed structures with one or several cell types. Basically, the nanoparticles allow it to glow and the group can image them with a camera. While this may not seem like a big deal, it can be a godsend for studying cells on the micro-scale. Biologists could also monitor oxygen distribution without any heavy equipment.

Professor Michael Kühl at the Department of Biology, University of Copenhagen explains: “3D printing is a widespread technique for producing the object in plastic, metal and other abiotic materials. Likewise, living cells can be 3D printed in biocompatible gel materials (bio-inks) and such 3D bioprinting is a rapidly developing field, eg, in biomedical studies, where stem cells are cultivated in 3D printed constructs mimicking the complex structure of tissue and bones. Such attempts lack online monitoring of the metabolic activity of cells growing in bio-printed constructs; currently, such measurements largely rely on destructive sampling. We have developed a patent-pending solution to this problem.”

Developing Biogels

The group developed the bio-ink by inserting glowing oxygen sensitive nanoparticles into the print matrix. When blue light excites the nanoparticles, they emit red luminescent light in proportion to the local oxygen concentration. With more oxygen, there is far less red luminescence and vice versa.

Cameras can capture the distribution of red luminescence and thus oxygen across bio-printed living structures. The main benefit is in online, non-invasive monitoring of oxygen distribution and dynamics that researchers can map to draw correlations with the growth and distribution of cells in the constructs without the need for destructive types of sampling.

Prof Kühl states that “it is important that the addition of nanoparticles doesn’t change the mechanical properties of the bio-ink, e.g. to avoid cell stress and death during the printing process.Furthermore, the nanoparticles should not inhibit or interfere with the cells.” Non-invasive forms of research are, after all, crucial to studying delicate forms of biology.

The research may even have further applications beyond the purely biological. It may be a great way of creating natural/non-electric luminescence in dark habitats or even rooms. It could be of great use in space, particularly with it’s ability to study oxygen levels without disturbing the samples. The research is still young and researchers are still experimenting with new forms.

Researchers in Korea have shown that graphene fibers can be reinforced with a mussel-inspired polymer. A research group led by Sang Ouk Kim at the Korea Advanced Institute of Science and Technology (KAIST) has utilized polydopamine as an effective infiltrate binder to produce graphene-based liquid crystalline fibers with impressive mechanical and electrical properties. The group reports its work in a paper in Advanced Materials.

This bio-inspired defect engineering approach is clearly distinguishable from previous attempts at employing insulating binders and offers great potential for producing materials for use in flexible and wearable devices, as well as low-cost structural materials. The two-step defect engineering approach addresses the intrinsic limitation of graphene fibers, which arises from their folding and wrinkling during the fiber-spinning process.

However, owing to the inherent formation of defects and voids caused by the bending and wrinkling of the graphene oxide layer within graphene fibers, their mechanical strength and electrical/thermal conductivities are still far below the desired ideal values. Finding an efficient method for producing densely packed graphene fibers with a strong interaction between the layers is thus a critical challenge.

Kim’s research group focused on the adhesion properties of polydopamine, a polymer inspired by the natural adhesive used by mussels, to solve the problem. This functional polymer, which is studied in various fields, can increase the adhesion between the graphene layers and prevent structural defects.

Using polydopamine as a binder, the group succeeded in fabricating high-strength graphene liquid crystalline fibers with controlled structural defects. They were also able to fabricate fibers with improved electrical conductivity by carbonizing the polydopamine.

Based on the theory that the high temperature annealing of polydopamine gives it a similar structure to graphene, the team optimized the dopamine polymerization conditions and showed that polydopamine could solve the inherent defect control problems of graphene fibers. They also confirmed that, compared with conventional polymers, polydopamine has improved electrical conductivity due to the influence of nitrogen in the dopamine molecules.

“Despite its technological potential, carbon fiber using graphene liquid crystals still has limits in terms of its structural limitations,” said Kim. “This technology will be applied to composite fiber fabrication and various wearable textile-based application devices.”

]]>3D ‘organ on a chip’ could accelerate search for new disease treatmentshttp://biomat.net/site2/news/3d-organ-on-a-chip-could-accelerate-search-for-new-disease-treatments/
Tue, 11 Dec 2018 21:58:57 +0000http://biomat.net/site2/?post_type=news&p=4673Original source: University of Cambridge

Researchers have developed a three-dimensional ‘organ on a chip’ which enables real-time continuous monitoring of cells, and could be used to develop new treatments for disease while reducing the number of animals used in research.

The device, which incorporates cells inside a 3D transistor made from a soft sponge-like material inspired by native tissue structure, gives scientists the ability to study cells and tissues in new ways. By enabling cells to grow in three dimensions, the device more accurately mimics the way that cells grow in the body.

The researchers, led by the University of Cambridge, say their device could be modified to generate multiple types of organs – a liver on a chip or a heart on a chip, for example – ultimately leading to a body on a chip which would simulate how various treatments affect the body as whole. Their results are reported in the journal Science Advances.

Traditionally, biological studies were (and still are) done in petri dishes, where specific types of cells are grown on a flat surface. While many of the medical advances made since the 1950s, including the polio vaccine, have originated in petri dishes, these two-dimensional environments do not accurately represent the native three-dimensional environments of human cells, and can, in fact, lead to misleading information and failures of drugs in clinical trials.

“Two-dimensional cell models have served the scientific community well, but we now need to move to three-dimensional cell models in order to develop the next generation of therapies,” said Dr Róisín Owens from Cambridge’s Department of Chemical Engineering and Biotechnology, and the study’s senior author.

“Three-dimensional cell cultures can help us identify new treatments and know which ones to avoid if we can accurately monitor them,” said Dr Charalampos Pitsalidis, a postdoctoral researcher in the Department of Chemical Engineering & Biotechnology, and the study’s first author.

Now, 3D cell and tissue cultures are an emerging field of biomedical research, enabling scientists to study the physiology of human organs and tissues in ways that have not been possible before. However, while these 3D cultures can be generated, technology that accurately assesses their functionality in real time has not been well-developed.

“The majority of the cells in our body communicate with each other by electrical signals, so in order to monitor cell cultures in the lab, we need to attach electrodes to them,” said Dr Owens. “However, electrodes are pretty clunky and difficult to attach to cell cultures, so we decided to turn the whole thing on its head and put the cells inside the electrode.”

The device which Dr Owens and her colleagues developed is based on a ‘scaffold’ of a conducting polymer sponge, configured into an electrochemical transistor. The cells are grown within the scaffold and the entire device is then placed inside a plastic tube through which the necessary nutrients for the cells can flow. The use of the soft, sponge electrode instead of a traditional rigid metal electrode provides a more natural environment for cells and is key to the success of organ on chip technology in predicting the response of an organ to different stimuli.

Other organ on a chip devices need to be completely taken apart in order to monitor the function of the cells, but since the Cambridge-led design allows for real-time continuous monitoring, it is possible to carry out longer-term experiments on the effects of various diseases and potential treatments.

“With this system, we can monitor the growth of the tissue, and its health in response to external drugs or toxins,” said Pitsalidis. “Apart from toxicology testing, we can also induce a particular disease in the tissue, and study the key mechanisms involved in that disease or discover the right treatments.”

The researchers plan to use their device to develop a ‘gut on a chip’ and attach it to a ‘brain on a chip’ in order to study the relationship between the gut microbiome and brain function as part of the IMBIBE project, funded by the European Research Council.

Quantum dots made from the carbon material graphene prevent alpha-synuclein from aggregating into strand-like structures known as fibrils. They also help disaggregate fibrils that have already formed. Alpha-synuclein fibrils are thought to be implicated in Parkinson’s disease because they kill dopamine-generating neurons, so the new findings might help in the development of therapies to treat this disease as well as others in which fibrilization occurs.

Synucleins are a family of proteins typically found in neural tissue. Researchers believe that one type of synuclein, alpha-synuclein, twists into fibrils, which then accumulate in the midbrain of patients with Parkinson’s. Treatments with efficient anti-aggregation agents might thus be one way of fighting the disease.

A team led by Byung Hee Hong of Seoul National University and Han Seok Ko of The Johns Hopkins University in Baltimore have now found that graphene quantum dots (GQDs) bind to alpha-synuclein in vitro. Thanks to fluorescence and turbidity assays, as well as transmission electron microscopy measurements, the researchers found that the dots prevent alpha-synuclein from forming into fibrils. The nanostructures also dissociate already-formed fibrils into short fragments, with the average length of the fragments shortening from 1 micron to 235 nm and 70 nm after 6 and 24 hours respectively. The number of fragments starts to decrease after three days too and cannot be detected at all after seven days, which implies that the fibrils completely disintegrate after this time.

Mice show improved symptoms of the disease after six months

In their experiments, Hong and Ko’s team also injected the GQDs into the bloodstream of transgenic mice with Parkinson’s and found that they showed improved symptoms of the disease after six months – as assessed by routine cylinder and pole tests. The mice showed fewer movement problems, were able to use both forepaws to balance themselves on cylinders and ran down poles quicker. The researchers say that these improvements could come from the fact that the quantum dots are small enough to penetrate the blood-brain barrier and protect against dopamine neuron loss induced by alpha-synuclein preformed fibrils.

The GQDs do not show any appreciable in vitro and in vivo toxicity after six months of “prolonged injection” either and can be cleared from the body and excreted into urine, they add. The quantum dots might produce a similar effect in other diseases in which fibrilization occurs. Indeed, previous research by another team has already shown that injecting them into mice with Alzheimer’s inhibits the fibrilization of beta-amyloid peptides.

Biomedical engineers from Duke University and Washington University in St. Louis have demonstrated that, by injecting an artificial protein made from a solution of ordered and disordered segments, a solid scaffold forms in response to body heat, and in a few weeks seamlessly integrates into tissue.

The ability to combine these segments into proteins with unique properties will allow researchers to precisely control the properties of new biomaterials for applications in tissue engineering and regenerative medicine.

The research appears online on October 15 in the journal Nature Materials.

Proteins function by folding, origami-like, and interacting with specific biomolecular structures. Researchers previously believed that proteins needed a fixed shape to function, but over the last two decades there has been a growing interest in intrinsically disordered proteins (IDPs). Unlike their well-folded counterparts, IDPs can adopt a plethora of distinct structures. However, these structural preferences are non-random, and recent advances have shown that there are well-defined rules that connect information in the amino acid sequences of IDPs to the collections of structures they can adopt.

Researchers have hypothesized that versatility in protein function is achievable by stringing together well-folded proteins with IDPs—rather like pearl necklaces. This versatility is obvious in biological materials like muscle and silk fibers, which are made of proteins that combine ordered and disordered regions, enabling the materials to exhibit characteristics like elasticity of rubber and the mechanical strength of steel.

IDPs are instrumental to cellular function, and many biomedical engineers have concentrated their efforts on an extremely useful IDP called elastin. A highly elastic protein found throughout the body, elastin allows blood vessels and organs—like the skin, uterus and lungs—to return to their original shape after being stretched or compressed. However, creating the elastin outside the body proved to be a challenge.

So the researchers decided to take a reductionist engineering approach to the problem.

“We were curious to see what types of materials we could make by adding order to an otherwise highly disordered protein,” said Stefan Roberts, a PhD student in the lab of Ashutosh Chilkoti and first author on the paper.

Due to the challenges of using elastin itself, the research team worked with elastin-like polypeptides (ELPs), which are fully disordered proteins made to mimic pieces of elastin. ELPs are useful biomaterials because they can undergo phase changes—go from a soluble to an insoluble state, or vice-versa—in response to changes in temperature. While this makes these materials useful for applications like long-term drug delivery, their liquid-like behavior prevents them from being effective scaffolds for tissue engineering applications.

But by adding ordered domains to the ELPs, Roberts and the team created “Frankenstein” proteins that combine ordered domains and disordered regions leading to so-called partially ordered proteins (POPs), which are equipped with the structural stability of ordered proteins without losing the ELPs ability to become liquid or solid via temperature changes.

Designed as a fluid at room temperature that solidifies at body temperature, these new biomaterials form a stable, porous scaffold when injected that rapidly integrates into the surrounding tissue with minimal inflammation and promotes the formation of blood vessels.

“This material is very stable after injection. It doesn’t degrade quickly and it holds its volume really well, which is unusual for a protein-based material,” Roberts said. “Cells also thrive in the material, repopulating the tissue in the area where it is injected. All of these characteristics could make it a viable option for tissue engineering and wound healing.”

Although the scaffold created by the POP was stable, the team also observed that the material would completely re-dissolve once it was cooled. What’s more, the formation and dissolution temperatures could be independently controlled by controlling the ratios of disordered and ordered segments in the biomaterial. This independent tunability confers shape memories on the POPs via a phenomenon known as hysteresis, allowing them to return to their original shape after a temperature cue.

The Duke team collaborated with the laboratory of Rohit Pappu, the Edwin H. Murty Professor of Engineering in the Department of Biomedical Engineering at Washington University in St. Louis to understand the molecular basis of sequence-encoded hysteretic behavior. Tyler S. Harmon, then a Physics PhD student in the Pappu lab, developed a computational model to show that the hysteresis arises from the differential interactions of ordered and disordered regions with solvent versus alone.

“Being able to simulate the molecular basis for tunable hysteresis puts us on the path to design bespoke materials with desired structures and shape memory profiles,” Pappu said. “This appears to be a hitherto unrecognized feature of the synergy between ordered domains and IDPs.”

Moving ahead, the team hopes to study the material in animal models to examine potential uses in tissue engineering and wound healing and to develop a better understanding of why the material promotes vascularization. If these studies are effective, Roberts is optimistic that the new material could become the basis for a biotech company. They also want to develop a deeper understanding of the interactions between the ordered and disordered portions in these versatile materials.

“We’ve been so fascinated with the phase behavior derived from the disordered domains that we neglected the properties of the ordered domains, which turned out to be quite important,” Chilkoti said. “By combining ordered segments with disordered segments there’s a whole new world of materials we can create with beautiful internal structure without losing the phase behavior of the disordered segment, and that’s exciting.”

Researchers have performed a careful comparison between locally generated, ischemia-induced, multipotent stem cells (iSCs) and bone marrow-derived mesenchymal stem cells (BM-MSCs) in an effort to determine which cell type has greater central nervous system (CNS) repair capacity. Their results show that the iSC characteristics make them more promising candidates as CNS injury therapeutics. The study is published in Stem Cells and Development, a peer-reviewed journal from Mary Ann Liebert, Inc., publishers. Click hereto read the full-text article free on the Stem Cells and Development website through November 26, 2018.

Takayuki Nakagomi, MD, PhD, with colleagues from the Hyogo College of Medicine and from the Kwansei Gakuin University School of Science and Technology, Hy?go, Japan, coauthored the article titled “Comparative Characterization of Ischemia-Induced Brain Multipotent Stem Cells with Mesenchymal Stem Cells: Similarities and Differences”. Although evidence has shown that grafted mesenchymal stem cells can improve neuronal function after a stroke, most of these cells never reach the target injured brain regions. However, a regional induction of stem cells occurs after ischemia that may provide greater opportunity to restore neuronal function. Thus, the authors of this study extracted iSCs from the ischemic regions of post-stroke mice and collected and prepared MSCs from bone marrow. They then compared the gene and protein expression, multipotency, and neuronal differentiation capacity of the two stem cell types. Ultimately, many similarities were identified between MSCs and iSCs, but only iSCs exhibited the potential for neuronal differentiation, thus establishing a case for their exploration as CNS therapeutic agents.

“Having recently demonstrated that ischemia-induced multipotent stem cells are present within the post-stroke human brain, the authors here seek to clarify how the potential of this fascinating cell population differs from that of mesenchymal stem cells.” says Editor-in-Chief Graham C. Parker, PhD, The Carman and Ann Adams Department of Pediatrics, Wayne State University School of Medicine, Detroit, MI.